Bone grafts and methods of making and using bone grafts

ABSTRACT

Provided herein are bone grafts and methods of making and using the same, as well as products and kits that include such bone grafts. In particular, bone grafts are provided that include osteogenic stem cells in a mix of osteoinductive demineralized bone matrix and osteoconductive cortico-cancellous chips.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.14/175,184, filed Feb. 7, 2014, which is hereby incorporated byreference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention generally relates to bone grafts, and methods ofmaking and using the same. More specifically, the present inventionrelates to osteogenic bone grafts that include osteogenic stem cells ina mix of osteoinductive demineralized bone and osteoconductivecortico-cancellous chips. Further included are kits and implants havingthe present bone grafts; and methods of making and using the presentbone grafts.

BACKGROUND

Bone generally has the ability to regenerate completely, e.g., after afracture but requires a very small fracture space or some sort ofscaffold to do so. Bone grafting is a surgical procedure that replacesmissing bone to repair bone fractures that are very complex, fail toheal properly, or pose a significant health risk to the patient.

Bone grafts may be autologous (bone harvested from the patient's ownbody, often from the iliac crest), allograft (cadaveric bone usuallyobtained from a bone bank), or synthetic (often made of hydroxyapatiteor other naturally occurring and biocompatible substances) with similarmechanical properties to bone. Most bone grafts are expected to bereabsorbed and replaced as the natural bone heals over a few months'time.

Bone grafts are osteogenic if they contain viable cells that are capableof bone regeneration. The current gold standard in bone graftsubstitutes for spine and long bone applications is autograft (i.e.,using the patient's own tissue), followed by allografts. Autografts areconsidered osteogenic, as they contain a high number of bone formingcells. However, autographs may have limited availability and they arelimited by donor site morbidity. Also, autografts may require multiplesurgeries. Allografts are limited by the large variability inperformance due to source and processing steps.

There is a need to produce superior bone grafts that are osteogenicand/or are able to enhance bone regeneration throughout the bone healingphase.

SUMMARY OF THE INVENTION

According to non-limiting example embodiments, the present inventionprovides bone grafts that include osteogenic stem cells in a mix ofosteoinductive demineralized bone and osteoconductive cortico-cancellouschips, to promote bone healing.

Other example embodiments are directed to methods for preparing the bonegrafts provided herein. Further example embodiments are directed tomethods that include administering a bone graft substitute to a mammalby surgically inserting one or more of the present bone grafts into amammal. The bone grafts may be administered for example by themselvese.g., in the form of a strip, putty, gel and sponge, or the bone graftmay be available in conjunction with an implant, such as beingincorporated therein or thereon.

Yet further example embodiments are directed to implants or otherdevices that include one more of the bone grafts provided herein thereinor thereon. Other example embodiments are directed to kits that includeone or more of the present bone grafts and/or components or ingredientsthat may be combined mixed or treated to prepare the present bonegrafts, as well as instructions, devices, implants, tools or othercomponents that may assist with making or using the present bone grafts.

BRIEF DESCRIPTION OF DRAWINGS

Non-limiting example embodiments are described herein, with reference tothe following accompanying Figures:

FIG. 1 is a flow chart of an example method of preparing a bone graftaccording to non-limiting examples of the present invention.

DETAILED DESCRIPTION

The present invention is drawn to bone grafts and methods for making andusing such bone grafts, as well as kits and implants or other devicesincluding the same.

While the example embodiments are described to be used in conjunctionwith healing bone fractures, it should be understood that these bonegrafts may be used for other purposes and therefore the presentinvention is not limited to such applications. In view of the teachingsprovided herein, one having ordinary skill in the art would recognizeother applications for which the bone grafts of the present inventioncould be used, and would be able to use the bone grafts and methods ofthe present invention in other applications. Accordingly, thesealternative uses are intended to be part of the present invention.

Additional aspects, advantages and/or other features of exampleembodiments of the invention will become apparent in view of thefollowing detailed description, taken in conjunction with theaccompanying drawings. It should be apparent to those skilled in the artthat the described embodiments provided herein are merely exemplary andillustrative and not limiting. Numerous embodiments of modificationsthereof are contemplated as falling within the scope of this disclosureand equivalents thereto.

In describing example embodiments, specific terminology is employed forthe sake of clarity. However, the embodiments are not intended to belimited to this specific terminology. Unless otherwise noted, technicalterms are used according to conventional usage.

As used herein, “a” or “an” may mean one or more. As used herein“another” may mean at least a second or more. Furthermore, unlessotherwise required by context, singular terms include pluralities andplural terms include the singular.

As used herein, the term “mammal” is intended to include any “subject”or “patient” animal, (including, but not limited to humans) to whom thepresent bone grafts may be administered. A subject or patient or mammalmay or may not be under current medical care, and may or may not havehad one or more prior treatments. As would be apparent to those skilledin the art, the formulations may be different for non-humans than forhumans.

As used herein, “an effective amount” refers to an amount of thespecified constituent in a composition or formulation, or an amount ofthe overall formulation that is effective in attaining results, thepurpose for which the constituent or composition is provided. Therefore,an effective amount of a bone graft formulation would be an amountsuitable for achieving the desired bone graft effect in a subject, suchas a mammal (e.g., human) to which the present bone graft isadministered.

Numerical data may be presented herein in a range format. It is to beunderstood that such range format is used merely for convenience andbrevity and should be interpreted flexibly to include not only thenumerical values explicitly recited as the limits of the range, but alsoto include all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited.

According to non-limiting example embodiments, the present inventionprovides bone grafts that include osteogenic stem cells in a mix ofosteoinductive demineralized bone and osteoconductive cortico-cancellouschips, for example to promote bone healing in a mammal. Exampleembodiments provide a bone graft material that is osteogenic (in whichliving bone cells in the graft material contribute to bone remodeling),osteoinductive (which encourages undifferentiated cells to become activeosteoblasts), and osteoconductive (which guides the reparative growth ofthe natural bone). Thus, example embodiments herein include bone graftsthat include (1) osteogenic stem cells and (2) a mix of osteoinductivedemineralized bone matrix and osteoconductive cortico-cancellous chips.

Non-limiting example embodiments also include methods of making thepresent bone grafts, which include (1) obtaining and/or preparingcortical chips by separating cortical bone from bone marrow, and rinsingand milling the cortical bone to chips with high length to width ratio;demineralizing the cortical chips e.g., in 0.5-0.7 N HCl for 15 min to 3hours; removing the HCl or other treatment; and rinsing with water untilthe pH of the water the chips are in is between 6.5 and 7; and (2)obtaining cancellous bone chips from fresh frozen condyles which may berinsed e.g., with saline; and further treating by one of the followingmethods:

-   -   (A) treating the cancellous chips with 0.3-0.5% saline, rinsing        the saline treated chips with phosphate buffered saline (PBS)        (e.g., 2 or more times), mixing the chips with freezing media        (e.g., minimum essential medium (MEM)) and 10% Dimethyl        sulfoxide (DMSO), and freezing the chips in media and storing at        temperatures between −80° C. and −180° C.;    -   (B) treating the cancellous chips with 0.3-0.5% saline, rinsing        the saline treated chips with phosphate buffered saline (PBS)        (2-3 times or more); culturing the chips (e.g., in minimum        essential medium), for up to 10 days; and after the culture        period mixing the chips with freezing media, and freezing the        chips in media and storing at temperatures between −80° C. and        −180° C.;    -   (C) treating the cancellous chips with collagenase (1 mg/ml-10        mg/ml) for 1-3 hours in an incubator set at 37° C. and 5% CO₂        with periodic agitation, forming a supernatant; filtering the        supernatant through a 70 micron cell strainer; centrifuging the        resulting cell suspension forming a cell pellet; reconstituting        the cell pellet in cell culture media and plating in tissue        culture flask; culturing the cells for up to 10 days e.g., at        37° C.; detaching the cells using a dissociation agent, such as        trypsin and reseeding on the cancellous chips to form cell        enriched cancellous chips; mixing the cell enriched cancellous        chips with freezing media and storing at temperatures between        −80° C. and −180° C.; or    -   (D) treating the cancellous chips with saline as in option (A)        above, and then treating and processing the chips with        collagenase as in option (C) above.

Non-limiting example methods according to the present invention aredepicted for example, in the flow chart of FIG. 1. As shown in FIG. 1,in example embodiments, methods of making the present bone grafts areprovided, which include obtaining and/or preparing cortical chips fromcortical shaft (see left side of flow chart) by removing bone marrow andrinsing in PBS, milling the cortical bone to chips with high length towidth ratio; demineralizing the cortical chips e.g., in 0.5-0.6 N HClfor 30 min -3 hours; and decanting HCl; and rinsing with water until thepH of the water the chips are in is between 6.5 and 7.

The right side of the flow chart of FIG. 1 shows the four different waysthat the cancellous bone chips of the present invention may be obtained.According to all of the options, cancellous bone chips are obtained fromfresh frozen condyles, which are milled to chips and treated withsaline. The chips are then further treated by one of the methods(Options A-D) set forth on the flow chart, which include:

-   -   (A) treating the cancellous chips with 0.3-0.5% saline, rinsing        the saline treated chips with phosphate buffered saline (PBS),        and freezing the chips in media at temperatures between −80° C.        and −180° C.;    -   (B) treating the cancellous chips with 0.3-0.5% saline, rinsing        the saline treated chips with phosphate buffered saline (PBS);        culturing the chips (e.g., in minimum essential medium) for up        to 10 days; adding freezing media, and freezing the chips in        media between −80° C. and −180° C.;    -   (C) treating the cancellous chips with collagenase (1 mg/ml-10        mg/ml) up to 3 hours; filtering supernatant and centrifuging to        obtain a cell pellet; reconstituting the cell pellet in cell        culture media for up to 10 days e.g., at 37° C.; and reseeding        the cell back on cancellous chips; or    -   (D) treating the cancellous chips with saline as in option (A)        above, and then treating and processing the chips with        collagenase as in option (C) above.

After the cortical chips and cancellous chips are obtained, thedemineralized cortical chips and cell enriched cancellous chips aremixed at a ratio of about 1:1 to 2:1. This last step is not depicted inFIG. 1. This method advantageously provides a bone graft material thatis osteogenic, osteoinductive and osteoconductive. According to exampleembodiments, the concentration of osteogenic cells in the bone graft maybe more than 20,000 cells/cc of final product.

According to example embodiments, the cortical bone chips may be milledto have a relatively high length to width ratio, for example having asize of e.g., 250 microns-3 mm. According to example embodiments, thecortical bone chips may be freeze-dried and stored at room temperature.

The cancellous chips in these embodiments may be frozen and stored at atemperature between −80° C. and −180° C., inclusive of the endtemperatures and ranges therebetween.

Methods of Use

Also provided herein are methods that include inserting any of thepresent bone grafts into a mammal in need of the bone graft. By way ofexample, the present bone grafts may be inserted into or administered toa mammal by surgically inserting one or more of the present bone graftsinto a mammal, such as a mammal in need thereof. The bone grafts may beinserted or administered for example by themselves e.g., in the form ofa strip, putty, gel and/or sponge, or the bone graft may be available inconjunction with an implant, such as being incorporated therein orthereon (e.g., as a coating). The bone grafts may be inserted in aneffective amount, as can be determined by a physician taking intoaccount the need for the bone graft, the type of bone graft, and thepatient.

As previously indicated, the subject/patient may be a mammal (as well asother animals), and the mammal may be (but does not have to be) human.

Embodiments of the present invention may include moldable and shapeableputty compositions that may be used for example to fill bone defects.Thus, according to example embodiments the present bone grafts may befor example in the form of a putty or other semi-solid or solid form,including, but not limited to, strip, putty, gel or sponge.

Implants

Yet further example embodiments are directed to implants or otherdevices or products that include one more of the bone grafts providedherein, incorporated into, or on the implant, or otherwise used with theproduct or implant. For example, the present bone graft substitutes maybe used as a graft within or inside an implant. By way of non-limitingexample, bone grafts may be used in conjunction with interbody spacersfor treatment of compression fractures.

Surgical implants and compositions should be biocompatible tosuccessfully perform their intended function. Biocompatibility may bedefined as the characteristic of an implant or composition acting insuch a way as to allow its therapeutic function to be manifested withoutsecondary adverse effects such as toxicity, foreign body reaction orcellular disruption. To help avoid adverse reaction, example bone graftsmay be prepared in sterile environments and formulations forimplantation into a mammal.

Kits

Yet further embodiments are directed to kits that include one or more ofthe present bone grafts or one or more components or ingredientsthereof.

Example kits may include for example, any of the present bone grafts,along with instructions and/or at least one additional component (suchas devices, implants, tools) that may be used for example in thestorage, preparation or use of the bone graft substitutes. By way ofexample, the kit components may be used to assist in adding the bonegraft to a device or implant, or to assist in inserting the bone graftinto a mammal. Further non-limiting examples may include one or more ofthe present bone grafts and instructions for the preparation of the bonegraft, instructions for the use of the bone graft, a tool for insertionof the bone graft into a mammal, a tool or vehicle for hydration of adry form of the bone graft, and/or an implant to be inserted into themammal with the bone graft. For example, the bone graft may be providedin a syringe for reconstitution and/or administration to amammal/patient. According to example embodiments, products may beprovided in a syringe with an attachment to deliver product in aminimally invasive manner. Other possible ingredients in kits mayinclude disposal implements or treatment literature.

Yet further non-limiting examples may include one or more ingredients ofthe present bone grafts, which may be combined, mixed or treated toprepare the present bone grafts. By way of example, the present kits mayinclude cortical and/or cancellous chips in any of the stages providedherein and/or other ingredients of the present bone grafts, which may becombined, mixed or treated in order to form the present bone grafts.Further provided may be instructions for preparation of one or more ofthe present bone grafts and/or one or more tools, devices, implants,and/or other components to assist in making or using the present bonegrafts.

The following example is provided to further illustrate variousnon-limiting embodiments and techniques. It should be understood,however, that these examples are meant to be illustrative and do notlimit the scope of the claims. As would be apparent to skilled artisans,many variations and modifications are intended to be encompassed withinthe spirit and scope of the invention.

EXAMPLES Example 1

This example demonstrates how to make example bone grafts that includeosteogenic stem cells in a mix of osteoinductive demineralized bone andosteoconductive cortico-cancellous chips, in accordance withnon-limiting example embodiments of the present invention.

The cortical and cancellous bones from long bones may be separated. Thecortical bone is separated from the bone marrow, rinsed in phosphatebuffered saline (PBS) solution and milled to chips (e.g., 250 microns-3mm size with a relatively high length to width ratio). The corticalchips may then be treated with 0.5 -0.7 N HCl, for 15 minutes—3 hours.At the end of the treatment, the HCl may be decanted and the chips maybe rinsed in deionized (DI) water until the pH is between 6.5 and 7. Thechips may then be freeze-dried and stored at room temperature.

The cancellous bone chips from fresh frozen condyles may be rinsed with0.9% saline 2-3 times. After the rinse, the chips may be treated in oneof the following ways:

(a) The cancellous chips may be treated with 0.3-0.5% saline. The salinetreated chips may then be rinsed in phosphate buffered saline (2 or moretimes). The chips may be mixed with freezing media (minimum essentialmedium (MEM)) and 10% Dimethyl sulfoxide (DMSO), and frozen between −80°C. and −180° C.

(b) The cancellous chips may be treated with 0.3-0.5% saline, followedby rinsing the chips in phosphate buffered saline (2-3 times). After therinse, the chips may be cultured in minimum essential medium, for up to10 days. At the end of the culture period the chips may be mixed withthe freezing media and frozen at a temperature between −80° C. and −180°C.

(c) The chips may be treated with collagenase (1 mg/ml-10 mg/ml) for 1-3hours at 37° C., with periodic agitation. At the end of the time, thesupernatant will be filtered through a 70 micron filter. The resultingcell suspension will then be centrifuged e.g., at 1000-1500 rpm for 5-15minutes. The cell pellet may be reconstituted in cell culture media andplated in tissue culture flask. The cells may be cultured for up to 10days. At the end of the time the cells may be detached using adissociation agent and reseeded on the cancellous chips. The cellenriched cancellous chips may then be mixed with freezing media andstored at a temperature between −80° C. and −180° C.

(d) the chips may be treated with saline as in option (a), and then betreated and processed with collagenase as in option (c). The finalproduct may be stored at between −80° C. and −180° C.

In all of the above cases, the concentration of osteogenic cells may bemore than 20,000 cells/cc of final product. The final bone graft productwill include demineralized cortical chips and cell-enriched cancellouschips that may be mixed at a ratio of about 1:1 to 2:1, inclusive of allpoints and ranges therebetween, including the end ratios.

The present invention provides a bone graft material that is osteogenic,osteoinductive and osteoconductive.

Example 2

This example exhibits another embodiment as to how to make example bonegrafts that include osteogenic stem cells in a mix of osteoinductivedemineralized bone and osteoconductive cortico-cancellous chips, inaccordance with non-limiting example embodiments of the presentinvention.

The cortical and cancellous bones from long bones (such as femur, tibia,radius and ulna) may be separated. The cortical bone is separated fromthe bone marrow, rinsed in phosphate buffered saline (PBS) solution andprocessed to produce chips (e.g., 250 microns-3 mm size) with arelatively high length to width ratio. The cortical chips/fibers maythen be treated with 0.5 -0.7 N HCl, for 15-40 minutes. At the end ofthe treatment, the HCl may be decanted and the chips may be rinsed indeionized (DI) water until the pH is between 6.5 and 7. The chips maythen be freeze-dried and stored at room temperature.

Condyles may be milled using a bone mill to produce cancellous chips inthe range of 0.05-1.5 mm. The cancellous bone chips from fresh frozencondyles are separated, for example by being rinsed with 0.9% saline 2-3times or more. After the rinse, the chips may be treated with 0.3-0.5%saline. The cancellous chips and the cortical fibers may be mixed in theratio of 1:1 and mixed with freezing media (Minimum essential medium)and 10% Dimethyl sulfoxide.

This final product may be stored at temperatures between −80° C. and−180° C.

In the foregoing specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the invention.Accordingly, it is intended that such changes and modifications fallwithin the scope of the present invention as defined by the claimsappended hereto. The specification and drawings are, accordingly, to beregarded in an illustrative rather than restrictive sense.

We claim:
 1. A bone graft comprising osteogenic stem cells, and a mix ofosteoinductive demineralized cortical fibers having a size in the rangeof 250 microns to 3 mm and osteoconductive cortico-cancellous chipshaving a size in the range of 0.05 to 1.5 mm, wherein the demineralizedcortical fibers and cortico-cancellous chips are mixed at a ratio of1:1.
 2. The bone graft of claim 1, wherein a concentration of theosteogenic stem cells in the bone graft is more than 20,000 cells/cc offinal product.
 3. The bone graft of claim 1, wherein the demineralizedcortical fibers are milled to a length to width ratio of 5:1 to 500:1.4. The bone graft of claim 1, wherein the demineralized cortical fibersare obtained by separating cortical bone from bone marrow, rinsing andmilling the cortical bone to fiber, and treating the cortical fibers toadjust the pH of the water the fibers are in to between 6.5 and 7 toform the demineralized cortical fibers.
 5. The bone graft of claim 1,wherein the cortico-cancellous chips are obtained from fresh frozencondyles and milling the condyles using a bone mill to produce thecortico-cancellous chips.
 6. The bone graft of claim 1 furthercomprising a freezing media and dimethyl sulfoxide.
 7. The bone graft ofclaim 1, wherein the bone graft is freeze dried.
 8. The bone graft ofclaim 1, wherein the bone graft is stored at a temperature between −80°C. and −180° C.
 9. A bone graft comprising osteogenic stem cells, and amix of osteoinductive demineralized bone matrix and osteoconductivecortico-cancellous chips.
 10. The bone graft of claim 9, wherein thedemineralized bone matrix and cortico-cancellous chips are mixed at aratio of about 1:1 to 2:1.
 11. The bone graft of claim 9, wherein aconcentration of the osteogenic stem cells in the bone graft is morethan 20,000 cells/cc of final product.
 12. The bone graft of claim 9,wherein the demineralized bone matrix is demineralized cortical fibers.13. The bone graft of claim 12, wherein the demineralized corticalfibers are milled to a length to width ratio of 5:1 to 500:1.
 14. Thebone graft of claim 12, wherein the demineralized cortical fibers areobtained by separating cortical bone from bone marrow, rinsing andmilling the cortical bone to fiber, and treating the cortical fibers toadjust the pH of the water the fibers are in to between 6.5 and 7 toform the demineralized cortical fibers.
 15. The bone graft of claim 9,wherein the cortico-cancellous chips are obtained from fresh frozencondyles and milling the condyles using a bone mill to producecortico-cancellous chips having a size in the range of 0.05 to 1.5 mm.16. The bone graft of claim 9 wherein the bone graft is freeze dried.17. The bone graft of claim 9, wherein the bone graft is stored at atemperature between −80° C. and −180° C.
 18. A method of promoting bonehealing in a mammal comprising inserting the bone graft of claim 9 intoa mammal in need of said bone graft.
 19. A product comprising an implantand at least one bone graft according to claim
 9. 20. A kit comprisingat least one bone graft according to claim 9 or at least one componentthereof, and at least one additional component selected from the groupconsisting of instructions for the preparation of the bone graft, one ormore tools, devices, or other components to assist in making or usingthe bone graft.